DJ-1 interacts with the ectopic ATP-synthase in endothelial cells during acute ischemia and reperfusion

Endothelial cells (ECs) play a central role in ischemia. ATP-Synthase is now recognized to be ectopically expressed in the cell surface of many cell types, with putative roles described in angiogenesis, proliferation, and intracellular pH regulation. DJ-1 is a multifunctional protein, involved in cell protection against ischemia, ischemia–reperfusion (I/R), and oxidative stress, that regulates mitochondrial ATP-synthase. Here we focused on the characterization of the endothelial dynamics of DJ-1, and its implication in the regulation of the ectopic ATP-synthase (ecATP-S) activity, during acute ischemia and I/R in ECs. We found that DJ-1 is secreted from ECs, by a mechanism enhanced in ischemia and I/R. A cleaved form of DJ-1 (DJ-1∆C) was found only in the secretome of ischemic cells. The ecATP-S activity increased following acute ischemia in ECs, coinciding with DJ-1 and DJ-1∆C secretion. The inhibition of DJ-1 expression inhibited the ecATP-S response to ischemia by ∼ 50%, and its exogenous administration maximized the effect, together with an enhanced Akt phosphorylation and angiotube-formation potential at reperfusion. Immunoprecipitation studies showed direct interaction between DJ-1 and the ecATP-S. Altogether suggesting that DJ-1 is actively cleaved and released from ischemic ECs and plays an important role in the regulation of the ecATP-S activity during acute ischemia and reperfusion.

Ischemia is defined as the stress resulting from the restriction of blood supply to a given tissue or organ. It is mainly caused by the capillary obstruction due to either microthrombus formation or microvascular damage. When this happens, oxygen unavailability disrupts the cellular metabolism leading to ATP depletion, acidosis, and the accumulation of detrimental products, ultimately resulting in an extensive cell death and organ dysfunction. Ischemia is a common hallmark of many diseases, as is the case of myocardial infarction, stroke, and kidney, limb, or intestinal ischemia, all of them responsible of a high morbidity and mortality worldwide. Furthermore, ischemia is also found in the core of most solid tumours, and is believed to play a role in the malignant cell progression 1 .
Endothelial cells (ECs), lining the circulatory system, play a pivotal role in regulating homeostasis and disease. Rather than a barrier, their key location between blood stream and the surrounding tissue, makes the endothelium an active player sensing and responding to hemodynamic changes and ischemic damage 2,3 . From normal embryonic development to tumour progression and the onset of an ischemic event, ECs provide multiple autocrine and paracrine signals supporting organ function 4 , and modulate the immune response 2 .
The F O F 1 ATP-synthase is the enzyme responsible for the formation of ATP from ADP and inorganic phosphate, driven by the electrochemical gradient established through the electron transport chain in the mitochondria. Despite it was originally believed to exclusively locate in the inner mitochondrial membrane, its ectopic expression in the cell surface have been proven for a wide spectrum of cell types, including vascular ECs 5-7 , hepatocytes 8,9 , adipocytes 10 , lymphocytes 11 , keratinocytes 12 , muscle 13 , and neural cells 14,15 , in both tumour and normal conditions 16 . It is known to act as a receptor for angiostatin 6 , and apolipoprotein A-I 7,8 , and to promote tumour-recognition by the immune system [16][17][18] . In ECs, ectopic ATP-synthase (ecATP-S) have been recognized to play a role in angiogenesis, proliferation, and regulating intracellular pH 7,19,20 . Furthermore, its location within the lipid rafts and caveolae makes plausible a functional connection with purinoreceptors, modifying the local

Results
Endothelial DJ-1 content declines during I/R. We investigated whether ECs subjected to I/R injury show a dysfunctional regulation of DJ-1 and/or its cleavage. Different periods of in vitro I/R were tested, and both the full-length and cleaved forms of DJ-1, as well as DJ-1 expression, were measured. A short period of reperfusion (i.e. 2 h) following ischemia led to a significant decline in intracellular full-length DJ-1 (p < 0.01; Fig. 1A-C). A significant effect was found in the interaction between duration of ischemia and duration of reperfusion (p < 0.05), meaning that DJ-1 decline at reperfusion was dependent on the severity of ischemia. Interestingly while after 2 h of reperfusion the levels of full-length DJ-1 were reduced from baseline (cells without ischemia), after 22 h of reperfusion, DJ-1 basal level was restored ( Fig. 1A-C). DJ-1∆C was found intracellularly at much lower levels than DJ-1 and although these levels seemed to rise at reperfusion, differences did not reach significance at initially measured time-points (Fig. 1B,C). The assessment of DJ-1∆C dynamics at different reperfusion times after 1 h of ischemia revealed that while DJ-1∆C intracellular content falls during ischemia, www.nature.com/scientificreports/ it increases with time after reperfusion (Supplemental Fig. 1). Regardless of the effects of I/R on DJ-1 protein content, no differences in DJ-1 gene transcription were found (Fig. 1D).
Acute ischemia and I/R promote DJ-1 and DJ-1∆C endothelial secretion. The reduced levels of DJ-1 in ECs after I/R may be explained by its release from the cell. Thus, we analysed the presence of DJ-1 forms in the secretome of ECs subjected to I/R. Interestingly, the full-length form of DJ-1 was detectable in the cell secretome in each of the conditions tested, with a significantly higher abundance after ischemia (p < 0.05) and after I/R (p < 0.01; Fig. 2), suggesting an active release of DJ-1 by cells under ischemic stress. Moreover, DJ-1∆C was found to be present just in the secretome of cells during ischemia alone (Fig. 2B) and occasionally during reperfusion, but never in the controls. These results suggest that DJ-1 is cleaved into DJ-1∆C and actively released by ECs during ischemia. In order to check whether DJ-1 and DJ-1∆C are secreted in a soluble form or within extracellular vesicles (EVs), we removed EVs by ultracentrifugation (1 h at 100.000 × g) of the supernatants collected after 1 h of ischemia prior protein precipitation. There were no differences on DJ-1 and DJ-1∆C levels ( Fig. 2B), indicating that both DJ-1 forms are found in the soluble secretome.

DJ-1 down-regulation impairs ectopic ATP-synthase activity rise after ischemia. Given that
DJ-1 has been previously reported to regulate the mitochondrial ATP-synthase activity 32 , and that ECs extracellular ATP generation increases after a period of incubation under ischemia-like conditions 22 , we sought to analyse whether ECs DJ-1/DJ-1∆C secretion under ischemia has an impact on the ecATP-S activity. Thus, we analysed the extracellular ATP generation of DJ-1 knocked-down ECs cultures exposed to normoxia and ischemia, compared to untransfected controls. After 1 h of ischemia, untransfected cultures exhibited nearly threefold increase in the extracellular ATP generation. Such increase after ischemia was significantly reduced in DJ-1 knocked-down cultures (p < 0.05; Fig. 3), indicating that protein DJ-1 is needed for the ecATP-S regulation after ischemia.
Extracellular DJ-1 boosts ectopic ATP-synthase activity after ischemia. In order to test whether the reported effect of DJ-1 upon the activity of the cell surface ATP-synthase is dependent on the extracellular form, we tested the extracellular ATP generation in ECs cultures subjected to ischemia or normoxia, in the presence and the absence of extracellular recombinant full-length DJ-1 and DJ-1∆C (at 100 nM). The administration www.nature.com/scientificreports/ of DJ-1 resulted in a highly significant increase of the extracellular ATP generation after ischemia (p < 0.01), independently of the DJ-1 form employed (Fig. 4A). This effect could be explained either by an increase of the ecATP-S activity or localization to the cell surface. After measuring the ectopic expression of the ATP-synthase following ischemia and I/R in the presence and the absence of exogenous DJ-1 and DJ-1∆C, no differences were detected across conditions ( Fig. 4B,C), meaning the reported changes in the extracellular ATP generation are a consequence of an activity increase rather than localization.

Extracellular DJ-1 interacts with the ectopic ATP-synthase. ECs were cultured for 1 h under
ischemic or normoxic conditions, in the presence and the absence of exogenous DJ-1 and DJ-1∆C. Thereafter, cells were thoroughly rinsed, lysed, and immunoprecipitated against ATP-synthase. Immunocaptures were then assayed by western blot for both ATP-synthase and DJ-1. While exogenous full length DJ-1 was detectable in both normoxic and ischemic cell immunocaptures, interaction with DJ-1∆C was only found in cells under ischemia (Fig. 5A,B). In order to check whether DJ-1/DJ-1∆C association with the ATP-synthase is preserved over the course of reperfusion, the same experiment was performed in cultures subjected to ischemia and reperfusion in the presence and the absence of DJ-1 and DJ-1∆C during the ischemic stimuli. As a result, after 2 h of reperfusion some traces of DJ-1∆C remained detectable in the ATP-synthase immunocapture, while the fulllength DJ-1 association with the ATP-synthase was lost. After 24 h of reperfusion, no association was detectable for DJ-1 of either form.

Extracellular DJ-1 enhances Akt phosphorylation and angiotube formation.
Because the extracellular ATP generation through the ecATP-S has been reported to activate downstream signalling involved in cell survival via purinoreceptors 20 . We analysed the Akt phosphorylation status in normoxia, ischemia, and I/R, in the presence and the absence of exogenous full-length DJ-1 or DJ-1∆C (at 100 nM). While no differences were found between treated and untreated cultures in normoxia or ischemia, an enhanced Akt activation during reperfusion was found for the cultures treated with DJ-1 in either form (p < 0.01; Fig. 6A,B). Within the endothelial cell, Akt is involved in a number of processes ranging from cell survival and inhibition of apoptosis to angiogenesis [36][37][38] . In order to test whether the Akt activation at reperfusion after the exposure to DJ-1 is involved in cell survival or angiogenesis, we analysed both the cleavage of Casp-3, as a surrogate of apoptosis, www.nature.com/scientificreports/ and tube-formation capacity at reperfusion, as a surrogate of the angiogenic potential. As a result, no cleavage of Casp-3 was detectable in any condition (Fig. 6C), but a significantly faster formation of capillary-like structures during reperfusion was seen for the treated cultures (Fig. 6D,E). The sub-lethal nature of the assayed ischemic protocol, proven by the absence of Casp-3 cleavage, further confirms that all reported effects following DJ-1 administration are indeed dependent on DJ-1 and independent of cell death.

Discussion
In the present study we characterized the endothelial dynamic of DJ-1 within the context of I/R injury, and explored its implications in the regulation of the ecATP-S activity following acute ischemia. We demonstrated that DJ-1 is actively cleaved and released by ECs, and depicted an autocrine effect upon the ecATP-S activity, extracellular ATP generation, and angiogenesis, in a human umbilical-vein endothelial cells (HUVEC) model of sub-lethal ischemia and I/R. Ever since their first isolation by Jaffe et al. back in 1973 39 , to current days, HUVECs have become a valuable model for the in vitro study of vascular physiology and disease. As a non-immortalized human cellular model of ECs, HUVECs faithfully reproduce human ECs behaviour, and have been largely employed to study a broad array of biological processes and diseases 40 . Consequently, there are a number of standardized assays for the analysis of endothelial physiology and angiogenesis in HUVEC, facilitating the comprehension and reproducibility of results.
It is widely accepted that protein DJ-1 is implicated in cell survival following I/R and oxidative stress, as previously proven both in vitro and in vivo by loss-of-function models of myocardial infarction and stroke 34,41-47 . www.nature.com/scientificreports/ However, although many molecular functions have been attributed to DJ-1, there is not yet a consensus on its exact molecular function. DJ-1∆C arises from a 15 amino acid carboxyl-terminal deletion of DJ-1, resulting from a specific cleavage in response to mild oxidative stress, and have been purposed as a mechanism triggering its cytoprotective activity 24,33,48 . We evaluated the impact of I/R upon the DJ-1 reservoir of ECs, and found a significant and ischemia-dependent decline of the full-length form after reperfusion. This observation may be explained by protein degradation or release to the extracellular space. We further demonstrated that both forms were present in the cell secretome. Interestingly, DJ-1∆C secretion was found to be associated to ischemia, suggesting that DJ-1 is actively cleaved and released by ECs during ischemia. Moreover, we demonstrated DJ-1 to be secreted in a soluble form (i.e. not bound to EVs), as the ultracentrifugation of the cell culture media prior sample processing had no effect upon the detected extracellular DJ-1 and DJ-1∆C. In order to avoid protein interference in the analysis of secretomes, we performed the secretion experiments under serum-free conditions, which may have affected the secretion process. Thus, two different controls were employed for ischemia and reperfusion, and so while both ischemia and first control underwent a transition from basal culture conditions to serum-free, both reperfusion and second control were kept in the absence of serum. Therefore, the observed differences in the DJ-1 secretion levels between the secretomes of the two controls arise from the acute cell stress induced by the transition from basal culture conditions to serum-free media.
Given the current consensus about a protective role for DJ-1, and the endothelial dynamic upon I/R injury 2,4,49 , the reported release of DJ-1 and DJ-1∆C may behave as a cell sensor for damage or as an autocrine/paracrine cell function modulator. Lacking of a conventional secretory signal peptide, DJ-1 has been previously suggested to be secreted through the autophagy-based unconventional secretion pathway, as proven by experiments with autophagy inhibitors and autophagy-related knockout models 50 . Being ischemia and I/R known stimuli to promote autophagy, this is could be a tentative and feasible mechanism. Yet, DJ-1 have been previously reported to be secreted under some pathologic conditions such as breast cancer 51 , Parkinson's disease 52,53 and stroke 54 , and a protective role upon ischemia 42 and I/R 55 have been proven for the extracellular form in neuronal cells. However, the mechanism by which extracellular DJ-1 confers protection remains to be clarified.
The ATP-synthase ectopic expression in the cell surface is now recognized for a number of cell types, and is known to display several functions ranging from angiogenesis to cholesterol uptake [6][7][8]21 . Being essentially a H + channel, a role in the regulation of the intracellular pH is presumable, as shown by its inhibition with monoclonal antibodies targeted to the ectopic ATP-synthase, which resulted in a dysregulation of the intracellular pH 20 . This mechanism would be especially relevant in ischemia, which rapidly leads to acidosis. Indeed, the ecATP-S has been reported to highly increase its activity under chronic ischemic conditions 22 , and has been proposed as a mechanism of ischemia tolerance for ECs 56 . Additionally, the ecATP-S has been proposed to affect downstream Despite not yet elucidated, its location within the lipid rafts and caveolae allows it to modify the local ATP/ADP concentrations, potentially inducing purinergic signalling 9,11,21 . Treatments with monoclonal antibodies targeted to the ectopic ATP synthase were shown to antagonize Akt and Erk1/2 signalling, and to activate JNK and MAPK-p38, in ECs 20 . Also, the accumulation of extracellular ATP have been reported to protect endothelial barrier integrity following I/R injury 57 . www.nature.com/scientificreports/ Here we report a great increase in the activity of the ecATP-S following acute ischemia in ECs, coinciding with DJ-1 and DJ-1∆C secretion. After 1 h of ischemia, control cultures exhibited nearly threefold increase in the extracellular ATP generation without changes in the relative amount of the cell-surface ATP-synthase. Such increase appeared to be dependent on DJ-1, as the inhibition of DJ-1 expression also inhibited the ecATP-S response to ischemia by ∼50%, and the administration of exogenous DJ-1 in either its full-length or cleaved form, maximized the effect. Noteworthy, the exogenous administration of DJ-1 in normoxia had no effect upon ecATP-S activity, possibly due to a lack of a proton gradient to drive its activity in the absence of acidosis. Hence, rather than activate the ecATP-S, extracellular DJ-1 seems to optimize its performance, as previously described in the mitochondria 32 . Furthermore, the exogenously administrated DJ-1 was proven to physically interact with the ATP-synthase in the same fashion seen for secretion. And so, interaction with ATP-synthase was found both under normoxia and ischemia for the full-length form of DJ-1, and exclusively under ischemia for the cleaved form. Such association was lost over the course of reperfusion in the absence of exogenous DJ-1.
Whilst ischemia represents a profound detrimental factor, with no other resolution than reperfusion, the rapid recovery of basal conditions paradoxically carries the potential to exacerbate damage itself in a process tightly correlated to the duration and severity of the ischemic insult 58 . Thus, despite being ischemia the most detrimental factor, the cell response to reperfusion directly impacts on the extent of damage 47 In the context of ischemia and I/R, Akt has a number of positives effects as is the case of the inhibition of apoptosis and the promotion of angiogenesis [36][37][38] . Several reports have shown an activation of Akt during early reperfusion after ischemia 59 . Furthermore, the extent of this activation inversely correlates with damage, and thus Akt have been proposed as a central element of the I/R injury and the so called reperfusion injury salvage kinase (RISK) pathway 60,61 . Interestingly, while the extracellular ATP generation through the ecATP-S has been reported to activate Akt signalling via purinoreceptors 20 , no activation of Akt was seen during ischemia in any condition. However, the ischemic exposure to DJ-1 in either form resulted in a more pronounced Akt activation at reperfusion, which may reflect a physiologic adaptation or an enhanced viability of ECs at reperfusion, rather than a consequence of purinergic signalling. A faster formation of capillary-like structures during reperfusion was seen for the treated cultures in the tube-formation assay, suggesting an enhanced angiogenic potential. Moreover, these reported effects were seen to be independent of cell death, as proven by the Casp-3 cleavage analysis, where no induction of apoptosis was found in any tested condition. Previous reports have proven a role for the ecATP-S in angiogenesis, as the treatment with targeted antibodies show an inhibitory effect 62,63 . Whether this effect is indeed dependent on the extracellular ATP generation, a secondary effect of cell pH regulation, or another feature conditional to the ecATP-S, remain to be clarified. Altogether, the data here reported supports a role for the ecATP-S and DJ-1 in the preservation of endothelial homeostasis in ischemia and I/R. Angiotube-formation assay. Tube-formation assay was performed as previously described 65 . Briefly, HUVEC were seeded in growth factor reduced MatriGel (Corning, New York, NY, USA) coated 48-well plates at a density of 30.000 cells/cm 2 , and let sit for 30 min. Then cultures were washed with PBS and subjected to 1 h in vitro ischemia in the presence and the absence of DJ-1 or DJ-1∆C at 100 nM. Immediately after, ischemia buffer was replaced with basal growth media (without DJ-1 and DJ-1∆C), and random acquisitions were taken every 2 h. Pictures were then analyzed in ImageJ (U.S. National Institute of Health, Bethesda, MD, USA).

Secretome analysis.
In order to avoid protein interference, analysis of the secretome was performed in serum-free conditions. HUVEC cultures were washed twice with PBS, and subjected to 1 h in vitro ischemia or kept in normoxia (1 h control). Immediately after, supernatants were collected and replaced by fresh medium. Cells were allowed to recover for 2 h. After such time, reperfusion and 2 h control supernatants were collected, and cells counted with a cell counter (Beckmann Coulter, Brea, California). Supernatants were centrifuged to discard detached cells and debris. Then, supernatants were either mixed with 5× radio-immune precipitation assay (RIPA) buffer supplemented with a protease inhibitior cocktail (05056489001; Roche Diagnostics, Man- Ectopic ATP-synthase activity. ecATP-S activity was analysed as previously described 19 . Briefly, cells were washed once in HEPES buffer (10 mM HEPES, 150 mM NaCl), and incubated for 5 min in HEPES buffer supplemented with 2 mM MgCl 2 . Extracellular ATP generation was then initiated with the addition of HEPES buffer supplemented with 2 mM MgCl 2 , 20 mM KH 2 PO 4 and 200 µM ADP. After 20 s of reaction, supernatants were collected in EDTA containing tubes at 5 mM final concentration and centrifuged at 300xg 10 min at 4 °C to discard detached cells. ATP concentration was then assayed in supernatants with a luminescent ATP detection assay kit (ab113849; Abcam, Cambridge, United Kingdom) following manufacturer's instructions. After supernatants collection, cells were immediately lysed and protein amounts were quantified to normalize between wells. Statistical analysis. Normality was assessed with the Shapiro-Wilk method. When normality could be assumed, statistical differences between groups were analysed by two-tailed t-test (for comparisons between two groups), one-way ANOVA (for multiple groups) or two-way ANOVA (for multiple groups and two factors). Tukey's Honestly Significant Difference (HSD) post hoc test was performed to correct significance for multiplecomparisons. Kruskal-Wallis rank sum test was performed when normality could not be assumed. Data is presented as mean ± SEM. All the analyses were performed in RStudio (RStudio, Boston, MA, USA).

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.